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Copyright 0 1983 by the Genetics Society of America
COMPLEMENTATION AND PRELIMINARY LINKAGE ANALYSIS
OF ZYGOTE MATURATION MUTANTS OF THE HOMOTHALLIC
ALGA, CHLAMYDOMONAS MONOlCA
KAREN P. VANWINKLE-SWIFTA N D CYNTHIA G. BURRASCANO
Deportment of Biology, San Diego State University, Son Diego, Colifornio 92282
Manuscript received September 27, 1982
Revised copy accepted November 17,1982
ABSTRACT
Sexual reproduction in Chlamydomonas monoica is homothallic: pair formation and cell fusion occur in clonal culture and give rise to a heavily walled
diploid zygospore. During maturation of the young zygote, a distinctive “primary
zygote wall” is released before the development of the highly reticulate zygospore wall. Using ethyl methanesulfonate and ultraviolet irradiation as mutagens, we have isolated 19 maturation-defective (zym) mutant strains which
upon self-mating produce inviable zygotes. These zygotes fail to release a
primary zygote wall, fail to develop the normal zygospore wall, and eventually
undergo spontaneous lysis. In nearly all cases, the mutations appear to be
expressed only in the diploid zygote; pleiotropic effects on vegetative cell
testing performed
growth or morphology are not evident.-Complementation
on 17 of these mutants indicates that all are recessive and that they define seven
distinct complementation groups. Preliminary tetrad analysis of two-factor and
multifactor zym crosses provides no evidence for physical clustering of the
maturation genes, and instead suggests that they are widely distributed throughout the nuclear genome.
exual reproduction in Chlamydomonas provides a potentially useful experS
imental system for the study of regulated gene expression during cellular
development. The sexual cycle of this unicellular eucaryotic alga comprises a
precise sequence of cellular events that must involve interactions between
regulatory genes and numerous structural genes expressed only in sexual cells
(i.e., gametes or zygotes). Progression of the sexual cycle involves the differentiation of vegetative haploid cells into sexually active gametes, the development
of a diploid zygote through fusion of compatible gamete pairs, the maturation
of the young zygote into an elaborately walled zygospore, entrance into and
maintenance of the dormant state, and, finally, breaking of dormancy and the
release of haploid asexual zoospores through meiotic germination of the zygospore in response to specific environmental conditions (LEVINEand EBERSOLD
1960).
Our present understanding of sexual differentiation in Chlamydomonas is
based primarily upon ultrastructural, biochemical, and genetic studies on the
heterothallic species, C . reinhardtii. In this species, the differentiation of a
vegetative cell into a sexually active form (gametogenesis) occurs in response to
Genetics 103 429-445 March, 1983
430
K. P.
VANWINKLE-SWIFT
AND
C. G.
BURRASCANO
nitrogen starvation (SAGER
and GRANICK
1954), and involves extensive intracellular changes (JONES, KATESand KELLER
1968; SIERSMA
and CHIANG
1971; MARTIN
and GOODENOUGH
1975; WEEKSand COLLIS1979). Differentiated gametes of
opposite mating-type differ from one another in terms of surface flagellar
1976; RAY,SOLTER
and GIBOR1978), and
proteins (BERGMANet al. 1975; SNELL
specialized mating structures (FRIEDMANN,
COLWIN
and COLWIN
1968; CAVALIERSMITH1975; TRIEMER
and BROWN1975; GOODENOUGH
and WEISS1975). These
flagellar differences promote agglutination between flagella of opposite mating
types, bringing the partners into close contact. Flagellar agglutination, in turn,
triggers the release of an autolytic enzyme facilitating shedding of the gametic
TAMAKI
and TSUBO1978)
cell wall (GOODENOUGH
and WEISS1975; MATSUDA,
and activates the mating structures, which upon contact produce a cytoplasmic
connection between the two cells promoting cell fusion (CAVALIER-SMITH
1975;
WEISS,GOODENOUGH
and GOODENOUGH
1977).
Genetic studies on sexual reproduction in C. reinhardtii have involved analysis of mutants defective in these early stages. Thus, mutants showing condi1975), as well as mutants with
tional gametogenesis (FORESTand TOGASAKI
specific flagellar defects preventing agglutination between opposite mating
HWANGand WARREN
types (GOODENOUGH
and JURIVICH 1978; GOODENOUGH,
1978) or interferring with normal cell fusion (GOODENOUGH
and WEISS 1975;
GOODENOUGH,
HWANGand MARTIN1976) have been described. Mutations linked
to the mating-type locus have been identified, whereas others are unlinked to
and TOGASAKI
1975;
mating type but show sex-limited expression (FOREST
GOODENOUGH,
HWANGand MARTIN1976; GOODENOUGH,
HWANGand WARREN
1978; HWANG,MONKand GOODENOUGH
1981).
Although genetic analyses of events unique to the diploid zygote have not
been reported for C. reinhardtii, this period in the sexual cycle is of obvious
interest to developmental geneticists. During the period of zygote development
(maturation), novel proteins appear, several of which are components of the
protective zygospore wall (MINAMIand GOODENOUCH
1978). Nuclear fusion
occurs early in zygote maturation, followed soon after by plastid fusion (CAVALIER-SMITH
1970, 1975; BLANK,GROBEand ARNOLD1978), and provides the
opportunity for recombination between parental DNAs. Dedifferentiation of the
zygotic plastid (CAVALIER-SMITH
1976) and selective methylation and degrada1979) also occur in the
tion of chloroplast DNAs (BURTON,
GRABOWY
and SAGER
maturing zygote. The highly resistant, albeit dormant, zygospore provides for
species survival under harsh environmental conditions which would be lethal
to vegetative cells (cf. LEWIN1951, and VANWINKLE-SWIFT
1977). Thus events
occurring after gamete fusion have major consequences in terms of species
survival and adaptability. These events appear to occur in a precise sequential
pattern suggesting a carefully regulated mechanism(s) for gene expression, and
most probably involve interacting signals initially received from the individual
parental gametes.
The apparent absence of zygote maturation mutants of C. reinhardtii undoubtedly reflects technical problems inherent in the use of an obligately
heterothallic species. In such species, the diploid zygote results only from the
MATURATION MUTANTS OF C. MONOICA
431
union of cells of stable opposite mating types, i.e., from interclonal matings.
Recessive mutations in genes expressed only in the diploid zygote cannot be
readily identified since their effects will be masked by the corresponding wildtype alleles in the heterozygous zygotes. In contrast, recessive mutations causing
defective zygote maturation should be easily detected in homothallic species
since homozygous mutant zygotes will be produced by self-mating within the
mutant clone. This point is well illustrated by the successful application of
homothallic yeast strains to genetic studies on yeast sporulation (analogous to
and EGEL
zygote maturation in Chlamydomonas) and meiosis (BRESCH, MULLER
1968; ESPOSITO
and ESPOSITO1974).
For these reasons, we have begun studies on the homothallic alga Chlamydomonas monoica (VANWINKLE-SWIFT
1979, 1980; VANWINKLE-SWIFT
and
BAUER1982). We report here the isolation of mutant strains (zym) exhibiting
abnormal zygote maturation. The responsible mutations are expressed only in
the diploid zygotes produced by self-mating within the mutant clones. Complementation testing has identified seven “cistrons” involved in zygote maturation,
and verifies that all of the maturation mutations analyzed are recessive lethals.
Preliminary tetrad analysis has provided no evidence for physical clustering as
a mechanism for coordinated expression of these maturation genes. A brief
and VANWINKLE-SWIFT
abstract of this work has been published (BURRASCANO
1982).
MATERIALS AND METHODS
Strains. The mutant strains of C. monoica utilized in this study were derived from the wild-type
strain maintained by The Cambridge Culture Collection and provided to us by Dr. RALPHA. LEWIN.
1979) and ger-1 (germinaThe origins of the spr-fd-1 (spectinomycin-resistant; VANWINKLE-SWIFT
tion-defective) mutant strains within which the maturation mutations (zym) were induced are
depicted in Figure 1.The ger-1 strain was originally isolated as a high efficiency mating subclone of
wild type and was only later found to be carrying the spontaneous mutation interfering with normal
zygote germination.
Culture conditions. Vegetative cultures of all strains were routinely grown on agar solidified
Bold’s basal minimal medium (BM) (BISCHOFFand BOLD1963) and were maintained under continuous cool white fluorescent light (3000-5000 lx) at 20-25’. To induce mating, vegetative cells were
transferred to a low phosphate-low nitrate liquid medium (LPN) as described previously (VANWINKLE-SWIFT
and BAUER1982) and were incubated at 19-21O under continuous illumination (30005000 Ix) for 3-7 days. Aliquots of 7-day-old LPN cultures containing mature zygospores were plated
on BM agar and were incubated in the dark for 1-3 days at ambient room temperature. At the end
of the dark incubation period, unmated cells were killed by inverting the plate over a dish of
chloroform for 15 sec, and the zygospores were induced to germinate by returning the plates to
continuous illumination.
Mutagenesis. The procedure for ethylmethanesulfonate (EMS) mutagenesis of vegetative cells of
the spr-fd-1 strain has been described previously (VANWINKLE-SWIFTand BAUER1982). For mutagenesis by ultravioltet irradiation (UV), vegetative cells of the ger-1 strain were removed from BM
agar plates and were resuspended in LPN mating-induction liquid medium (CO. IO6 cells/ml). The
culture was maintained under continuous illumination (3000 lx) for 18 h r after which 10-ml aliquots
were removed and placed in small sterile Petri dishes. The aliquots were stirred continuously and
were irradiated using a General Electric 30W germicidal UV lamp held at 10 cm from the surface of
the cultures. After irradiation for 0,30, 60 or 90 sec, the cultures were placed in darkness for 6 h r to
prevent photoreactivation of the UV-induced damage. At the end of the dark incubation period,
several 0.03-ml aliquots from each UV dose were plated separately on BM agar at 0, 0.1 and 0.01
432
K. P. VANWINKLE-SWIFT A N D C. G. BURRASCANO
Cmonoicu
spont.
__I
ger-l
I
(Cambridge 11/70)
I
FdURD
1
uv
spr - fd /
EMS
f6-2/)
spr-fd / zymo-5)
I
X
fspr-fd /'zym-3'ger-/+zym-6"/
FIGURE1.-Derivation of strains. Abbreviations: FdURD = 5-fluorodeoxyuridine; UV = ultraviolet irradiation; EMS = ethyl methanesulfonate; spont. = spontaneous mutation; spr-fd-l = high
level spectinomycin resistant mutant (VANWINKLE-SWIFT
1980); ger-1 = germination-defective
mutant; zym = zygote maturation-defective mutants; wtl5c = wild-type recombinant tetrad product
from the cross: spr-fd-l zym-3 x ger-1 zym-6.
dilutions. The plated cells were allowed to grow for 7-10 days under standard conditions. Dilution
plates were used to assay plating efficiency and percent survival in the same way as previously
and BAUER1982). Cells derived from the 30-sec
described for EMS mutagenesis (VANWINKLE-SWIFT
UV dose (yielding 28% survival) were chosen for the mutant search. The cells obtained after postmutagenesis growth of the undiluted aliquots were removed from the BM plates and suspended in
10 ml BM liquid in standard test tubes. The tubes were illuminated from above for 30 sec after
which the top few milliliters, containing motile phototactic cells, were removed and streaked onto
several BM agar plates to permit isolation of individual post-mutagenesis progeny clones. From 200
to 400 clones derived from each of four aliquots of the original mutagenized culture were picked
and transferred to BM agar for 5 days further growth before screening for mutants.
Identification of zym mutants. The methods for screening for zygote maturation mutants (zym)
have been described previously (VANWINKLE-SWIFT
and BAUER
1982).Briefly, each post-mutagenesis
progeny clone was suspended separately in a 0.3-nil aliquot of LPN mating-induction liquid medium,
incubated under continuous illumination at 21' for 3-4 days, and then sampled for inspection by
phase contrast microscopy. For a wild-type strain, a 3- to +day incubation in LPN liquid medium
is sufficient to allow for gametic differentiation, pair formation, cell fusion, release of a primary
zygote wall, and nearly complete development of the highly reticulate zygospore wall. The LPN
cultures of post-mutagenesis progeny clones were assayed for the presence of the discarded primary
MATURATION MUTANTS OF C. MONOICA
433
zygote wall and for zygote morphology. Additional details of the screening procedure can be found
in the text or in VANWINKLE-SWIFT and BAUER 1982. Maturation mutants (zym) were assigned
sequential identification numbers indicating their order of isolation. Because of possible complications in the interpretation of complementation data (see DISCUSSION),
renaming of the mutants to
indicate locus position is not yet warranted.
Complementotion analysis. Visual inspection: The maturation-defective mutant clones were
suspended in LPN medium in all possible pairwise combinations and were allowed to undergo
gametogenesis, mating and zygote maturation (4days). Clonal cultures of each parental strain were
set up at the same time to verify that each strain had become sexually active. Aliquots were
removed from each pairwise culture and inspected by phase contrast microscopy to determine
whether normal, fully matured zygospores were produced (by crossing) in addition to the maturation-defective zygotes resulting from continued self-mating within each strain. The presence of
normal fully matured zygospores and discarded primary zygote walls in a mixed culture was taken
as an indication that the parent strains carried recessive complementary zym mutations. See text
for further discussion. Chloroform selection: Each UV-induced zym mutant was incubated in LPN
induction medium with the EMS-induced zym-1 strain. After 7 days the culture was plated on BM
agar and placed in darkness for 1-3 days. The plates were then removed from the dark, inverted
over a dish of chloroform for 10-20 sec and then placed under continuous illumination for 7-10
days. Growth after chloroform treatment indicated the presence of normal fully matured zygospores
in mixed cultures and thus served to identify UV-induced zym mutants complementary to zym-1.
Application of the chloroform selection method for complementation analysis to pairwise cultures
of UV-induced mutants could not be undertaken until the ger-l marker had been removed from
each of the UV-induced zym strains via tetrad analysis of EMS-zym X UV-zym crosses (see below).
Tetrad analysis. The procedures for maturation of zygotes, induction of zygote germination and
dissection of tetrads have been described previously (VANWINKLE-SWIFT
and BAUER1982). Crosses
between the EMS-induced zym-1 strain (carrying the spr-fd-l marker) and each complementary
UV-induced zym strain were performed and, whenever possible, 20-30 complete tetrads were
isolated. Each tetrad product was scored for spectinomycin resistance by transfer to minimal
medium supplemented with 40 pg/ml spectinomycin, and for zym marker genotype by inspection
of 4-day-old LPN cultures containing each tetrad product singly, as well as mixed cultures containing
the tetrad product and each of the parental zym strains (i.e., zym marker composition was
determined by complementation testing of each tetrad product). Tetrads were classified as parental
ditypes (PD), nonparental ditypes (NPD), or tetratypes (T) with respect to each possible marker pair
and linkage was assayed by the PD:NPD ratio in each case.
A small random sample of single UV-induced and double zym mutant tetrad products from each
of these initial crosses was scored for the presence of the ger-l marker by crossing each product to
a complementary UV-induced zym mutant carrying ger-1. The resultant zygotes were matured and
induced to germinate following standard procedures. Successful germination, as evidenced by
growth after chloroform selection, indicated that the tetrad product did not carry the ger-l marker.
Germination-proficient tetrad products carrying each of the UV-induced zym markers alone and in
combination with zym-l were saved for use in further constructions of multiply marked strains.
The procedures for the analysis of multifactor zym crosses were identical to those described above
for two-factor zym crosses.
Centromere-linked markers were identified as those which in combination yielded a low
frequency of tetratype tetrads. Gene-to-centromere distances (percent recombination or map units)
for other markers were then estimated as one-half the tetratype frequency obtained for the new
1965; HAWTHORNE
and
marker and a centromere-linked marker included in the cross (cf. GOWANS
1960). Alternatively, for multifactor crosses in which three markers segregated indeMORTIMER
pendently of one another, centromere distances to each of these markers were calculated from the
various tetratype tetrad frequencies according to the formulae developed by WHITEHOUSE
(1950; see
also GOWANS
1965).
RESULTS
ldentification of zygote maturation-defective (zym) mutant strains. Zygotes
produced by self-mating within individual post-mutagenesis clones recovered
K.
434
P. VANWINKLE-SWIFT AND C. G. BURRASCANO
by EMS or UV mutagenesis were examined by phase contrast microscopy. In
a standard mating, pair formation occurs within 36 hr after inoculation of
nutrient-limited liquid medium (LPN) with vegetative cells. At the end of a 3day incubation, most zygotes are fully rounded and a distinctive “primary
zygote wall” has been released into the culture medium. We have used the
presence of this discarded primary zygote wall as an indicator of normal zygote
maturation in clonal cultures. As summarized in Table 1, observations on 3700
individual clonal mating cultures revealed 19 maturation-defective mutants.
The majority of these mutants exhibit the same general phenotype: failure to
release a primary zygote wall, a tendency to produce zygotes that are not
uniformly spherical, extensive bleaching of the zygote, and eventual disintegration of the immature zygote without development of the reticulate zygospore
wall. In addition to this general phenotype, the zym-4 and zym-17 mutants
show a reduction in self-mating efficiency, and the latter strain also exhibits a
reduced vegetative growth rate. In contrast, the zym-16 and zym-18 mutants
(which may in fact be duplicates of the same original mutation; see Table 1) are
less stringent: apparently mature zygospores and discarded primary zygote
walls appear at a low frequency in clonal LPN cultures of these strains, although
maturation-defective zygotes are more common.
Complementation analysis. Interstrain matings between mutant strains carrying complementary zym markers result in normal fully matured zygospores
TABLE 3
Recovery of maturatioIi-defective (zym) mutants after induced mutagenesis
Mutagen
50 mM EMS
% Survival
100
(90min)
100 mM EMS
(60 min)
100 mM EMS
(90 min)
30 sec UV
(6 hr dark)
37
25
28
Zvm mutants
Aliquot No. clones
A
B
C
D
E
137
120
155
A
B
C
D
E
230
248
120
120
120
A
B
C
D
E
368
120
170
zym-3
zym-2
zym-1
zym-4
178
247
70
146
zym-5
A
B
300
240
C
D
300
360
zym-10
zym-6, zym-7, zym-9, zym-11, zym-13, zym20, zym-21
zym-14, zym-15, zym-16, zym-17, zym-18
zym-8
MATURATION MUTANTS OF C. MONOICA
435
(and release of the primary zygote wall). Pairwise mixed mutant cultures will
thus contain normal zygotes (zygospores) as well as maturation-defective zygotes (resulting from continued intrastrain pairings within the mixed cultures)
if the mutants carry recessive and complementary markers, but will contain
only maturation-defective zygotes if either marker is dominant or if the two
mutations are within the same cistron. Typical observations for a pair of
complementary zym strains are illustrated in Figure 2. The results of observations on all pairwise combinations of 17 zym strains are summarized in Figure
3 and define seven cistrons affecting zygote maturation.
As is typical of Chlamydomonas species, the normal mature zygospore of C.
monoica is resistant to brief exposure to acetone or chloroform vapor, whereas
unmated gametes and vegetative cells are not (LEWIN1951). Zym zygotes, if
plated on standard medium before their spontaneous lysis in liquid LPN cultures, do not germinate although they remain intact for at least several days.
Thus zym zygotes do not produce viable progeny whether or not chloroform
FrcuRe 2.-Phase contrast microscopy
.. of in1 I- and inter-strain mated cultures of the complementary zym-6 and eym-13 maturation mutants. (a) clonal w115c culture: (b) clonal zym-6 mutant
culture: (c) clonal zym-13 culture: (d)mixed culture containing both the zym-6 and zym-13 strains.
Abbreviations: Z = normal fully matured zygospore: YZ = young immature zygote: W = discarded
“primary zygote wall”; M = maturation-defective zygotes. Scale Bar = 10 pM.All observations are
on cultures maintained in mating-induction medium (LPN) for 4 days. The smallcr size of the zym6 and zym-13 zygotes is a consequence of a pleiotropic effect of the ger-1 marker present in these
strains (see Figure 1 ) and affecting both vegetative haploid and zygotic cell size.
436
K. P. VANWINKLE-SWIFT AND C. C. BURRASCANO
1 2 3 5 6
I
2 ...
0 9 10 I I 13 14 15 16 I8 2 0 2
I
?
4
-
3 ......
5
.........
6
............
7
...............
8
...............
9
...............
IO
...............
11
...............
13
............
L
14
I
...............
15 . . . . . . . . . . . . . . .
16
...............
18
...............
20
.............................................
21
4
I
................................................
A
B
C
0
E
F
G
- - w - - - 1,2,3,5,10,
6.9
7
14,15,21
8
II, 13
16,18
20
RCURE3.-Complementation analysis of maturation-defective mutants. Numbers correspond to
the I.D.numbers of the various zym mutants. Shaded squares indicate complementary pairs of
mutant strains which produced normal zygospores in mixed cultures: open squares show mutant
combinations for which marker complementation was not observed. Zym strains excluded from
and BAUER1982) to be
this analysis include the zym-4 strain, previously found (VANWINKLE-SWIIT
noncomplementary to the other EMS-induced zym strains (zym’s 1-3, 5). but which shows such
poor mating efficiency that accurate testing against the UV-induced mutants has not been possible,
and the zym-17 mutant which shows similarly poor mating. Strains initially designated zym-I2 and
zym-19 were discarded as unstable “phenocopies” which later showed normal zygote maturation.
selection has been employed. However, unmated zym haploids (vegetative cells
or gametes) remain viable for prolonged periods in mating-induction medium,
and chloroform exposure after plating is necessary to select heterozygous
zygospores. Complementation analysis is readily accomplished by exposing
plated aliquots from mixed mated cultures to chloroform and assaying for
MATURATION MUTANTS OF C. MONOICA
437
subsequent growth (indicating the presence of fully matured heterozygous
zygospores carrying complementary zym markers).
The application of this technique was initially hampered by the presence of
the spontaneous ger-1 mutation (a recessive mutation that allows normal zygote
maturation but blocks subsequent germination of the zygospore) in all of the
AND METHODS,
Fig. 1).Thus
UV-induced zym mutant strains (see MATERIALS
the chloroform selection assay for complementation could be applied initially
only to mutant combinations including one EMS-induced zym strain (germination-proficient) and one UV-induced zym strain (germination-defective), as
illustrated in Figure 4. Removal of the ger-2 marker through tetrad analysis of
these two-factor zym crosses (see below) eventually allowed us to use the
chloroform selection assay to verify all of the original complementation data
(Fig. 3) obtained by visual inspection of mixed cultures.
FIGURE
4.-Chloroform selection assay for complementation analysis of maturation-defective
strains. The wild-type vegetative cells and zygotes are derived from strain wtl5c (see Figure 1).The
aliquot of vegetative cells was taken from a standard liquid culture (asynchronous): all other
aliquots were taken from 7-day-old LPN cultures containing a high proportion of zygotes (normal
or maturation-defective). See MATERIALS
AND METHODS,
and text, for further details. Growth within
the central spot-derived from a mixed zym-l/zym-6 mating exposed to chloroform-indicates
complementation between zym-1 and zym-6. The same result was obtained after the ger-1 marker
had been removed from the zym-6 strain (not shown).
438
K . P. VANWINKLE-SWIFT AND C. G. BURRASCANO
Tetrad analyses. To determine linkage relationships between the UV-induced
zym mutations, and to permit construction of multiply-marked mapping stocks,
it was first necessary to remove the ger-2 mutation from the UV-induced
mutants. This was accomplished by crossing each to the EMS-induced zym-2
strain (which also carried the additional Mendelian marker, spr-fd-2, conferring
resistance to the antibiotic spectinomycin). The ger-2 marker was scored (see
AND METHODS)
in a small number of randomly selected zym tetrad
MATERIALS
products (parental zym products and double zym recombinants) to allow
recovery of germination-proficient mapping stocks, but no attempt was made to
determine the linkage relationships between ger-2 and the various zym loci.
From these preliminary crosses, complete tetrads were analyzed for progeny
genotypes with respect to the spr-fd-2, EMS-induced zym-2, and the various
UV-induced zym markers (Table 2). Although evidence was obtained initially
for linkage between zym-2 and zym-13 (Table 2 ) , analysis of subsequent multifactor crosses (Table 3) failed to support this conclusion. The earlier interpretation was also contradicted by the apparent absence of linkage between zym1 and zym-22 which maps to the same cistron as zym-13 by complementation
analysis (Table 1,Figure 3).
Although sample sizes are too small to rule out very loose linkage, the data
from these preliminary crosses provide no evidence for tight linkage between
zym-2 and any of the UV-induced zym markers, or between spr-fd-2 and any of
the zym loci. However, analysis of multifactor crosses suggests that zym-6 and
zym-8 are loosely linked since we have found a consistent excess of parental
ditype tetrads relative to nonparental ditypes in all crosses involving these
TABLE 2
Tetrad analysis of crosses of the general type: spr-fd-1 zym-1 X ger-1 UV-zym
Marker pairs
zym-VJV-zym
UV-zym
P
NP
zym-6
zym-7
zym-8
zym-9
zym-11
zym-13
zym-16
zym-18
zym-20
7
3
8
5
5
6
10
8
4
8
13
18
3
2
8
11
5
15
T
7
11
13
7
5
9
8
4
12
spr-fd-l/zym-l
spr-fd-l/UV-zym
PNP
P
NP
T
P:NP
P
NP
T
P:NP
0.88
0.60
0.57
1.33
1.30
2.25*
0.27
0.40
0.53
5
5
13
7
3
4
7
13
4
4
5
0.38
0.71
2.67
0.42
1.00
6
6
1.58
12
15
8
2.25
7
8
9
9
5
7
4
9
10
0.86
1.00
1.38
5
13
7
6
8
9
12
5
0.20
4
2
5
2.00
12
0.92
7
13
10
0.54
71
80
68
0.89
8
5
13
19
9
1
11
12
13
13
4
5
12
11
0.56
1.08
0.80
0.88
Abbreviations: P = parental ditype tetrad; NP = non parental ditype; T = tetratype tetrad; UVzym = zym mutant induced by UV irradiation of the ger-1 strain (see Figure 1).* Indicates that the
excess of parental ditype tetrads is statistically significant (p = 0.05). For a given cross, the numbers
of tetrads reported for the various marker pairs are not always equal because of occasional
ambiguities in scoring a particular marker.
439
MATURATION MUTANTS OF C. MONOICA
markers, and a tetratype:nonparental ditype ratio exceeding 4:l (Table 3; see
discussion by GOWANS
1965).
Tetrad analysis also revealed that three of our markers-spr-fd-1, zym-6 and
zym-13-may be relatively close to their respective centromeres since in pairwise combinations these markers yield low tetratype frequencies (Tables 2, 3).
By including these “centromere-linked markers” in crosses we can estimate
other gene-to-centromere distances as one-half the tetratype frequency for the
centromere-linked marker and the second marker under analysis. This assumes
that no crossing-over occurs between the centromere and the gene being used
as a centromere-marker (see GOWANS
1965 for discussion).
Alternatively, the formulae derived by WHITEHOUSE
(1950), which utilize
tetratype tetrad frequencies from crosses involving three independently segregating markers, can be applied to estimate gene-to-centromere distances for
several gene loci. The derivation of these formulae has been discussed in detail
by GOWANS(1965). Table 4 compares the gene-to-centromere distances for
several zym gene loci calculated by the various methods. These distances have
been calculated and compared according to the data from the multifactor
crosses of Table 3 in which the tetratype tetrad frequencies for the spr-fd-I/
zym-6 marker combination is somewhat higher than we typically observe.
However, analysis of this marker combination in a total of 14 crosses yielding
a pooled sample of 280 tetrads (unpublished data) gives a tetratype frequency
of 0.08. Thus we feel application of methods assuming centromere linkage for
the spr-fd-1 or zym-6 loci (methods A1 and AZ, Table 4) can provide reasonably
accurate estimates of gene-to-centromere distances for other markers. Although
there is relatively close agreement between the independent estimates for a
given gene-to-centromere distance (Table 4), the differences in centromere
TABLE 3
Pooled data from tetrad analysis of multifactor zym crosses
~~
P
Marker pair
~~
spr-fd-l/zym-l
spr-fd-l/zym-6
spr-fd-l/zym-8
spr-fd-l/zym-13
zym-l/zym-6
zym-l/zym-8
zym-l/zym-13
zym-6/zym-8
zym-6/zym-13
zym-8/zym-13
NP
T
PNP
T:NP
%T
24
28
15
13
23
0.83
0.96
0.53
0.96
1.38
0.23
1.22
3.64
0.60
0.34
0.14
0.64
0.09
0.35
0.69
0.26
0.71
0.03
0.79
~
20
27
8
18
22
7
10
14
15
3
9
41
3
18
11
22
1.22
40
0.64
15
5
17
4
9
0.67
2.80*
0.88
0.75
46
1
26
0.32
2.73
9.20**
0.06
6.50
Crosses analyzed: spr-fd-1 zym-6 x zym-1 zym-8; spr-fd-1 zym-1 x zym-6 zym-8 zym-13; spr-fd1zym-6 zym-8 X zym-1 zym-7; spr-fd-l zym-1 zym-20 X zym-6 Zym-8 zym-13.
Abbreviations: P = parental ditype tetrad; NP = nonparental ditype; T = tetratype tetrad.
* Statistically significant (p = 0.05) excess of parental ditype tetrads relative to nonparental ditypes
suggests linkage. * * T:NP ratio statistically greater (P = 0.05) than 4:l provides additional support
for hypothesis of loose linkage between zym-6 and zym-8. Because of smaller pooled sample sizes,
for the zym-7 and zym-20 markers, data for these loci have been excluded from this analysis.
440
K . P.
VANWINKLE-SWIFT
A N D C. G. BURRASCANO
TABLE 4
Estimated centromere-to-gene distances (map units)
Centromere (C)-to-gene interval
Method
C1-spr-fd-1
A. 1.
C2-zym-6
2.
0"
7.0
0"
4.5
1.5
3.
4.5
1.5
0"
3.3
4.2
B. 1.
2.
(i)
(ii)
(iii)
7.0
C3-zym-13
C4-zym-1
17.2
17.7
13.2
C2-zym-8
32.0
35.4
39.4
14.5
b
b
1.3
35.8
35.4
31.9
Methods: A = use of "centromere-linked markers", including spr-fd-1 (A. I.), zym-6 (A. 2,), or
zym-23 (A. 3.); B = application of the formulae derived by WHITEHOUSE
(1950) using tetratype tetrad
frequencies for crosses in which three markers (spr-fd-1, zym-1, and zym-6) are segregating
independently (B. l.),or simplified by incorporating a previously determined (by Method B. 1.)geneto-centromere distance (see GOWANS1965) such as C4-zym-I (B. 2. i.), C2-zym-6 (B. 2. ii.), or C1spr-fd-l (B. 2. iii.). All estimates are based on data from Table 3.
a Value is assumed by the method.
* Negative values were obtained.
distances obtained for the several zym loci provide further evidence that the
maturation genes are not tightly clustered, but rather reside at well-separated
sites within the nuclear genome. A complete description of the linkage groups
involved will require continued mutant searches and more extensive analyses
of additional multifactor crosses (in progress).
DISCUSSION
The successful isolation of maturation-defective mutants of C. monoica
verifies our initial assumption that this self-mating species could provide a
unique opportunity for the identification of recessive mutations in genes expressed only in the transient diploid stage (i.e., the zygote) of the Chlamydomonas life cycle. The identification of seven complementation groups within a
sample of 17 mutant strains analyzed suggests that many genes are required for
normal zygote development and that mutations in several genes can confer an
identical maturation-defective (zym) phenotype. Preliminary genetic analyses
of multifactor crosses reported here, as well as additional unpublished data,
further suggest that these maturation genes are scattered throughout the nuclear
genome, since no conclusive evidence for tight linkage between the zym markers
has yet been found. Although no recombination has been detected between the
complementary zym-13 and zym-20 markers (data not shown), we will present
evidence elsewhere that the zym-20 mutant carries a major chromosomal
aberration (translocation or inversion) because meiotic lethality is common in
crosses involving this strain. Thus, the apparent linkage between zym-13 and
zym-20 may be an artifact created by selection against recombinant meiotic
products.
MATURATION MUTANTS OF C. MONOICA
441
Our two mutagenesis experiments yielded very different results both in terms
of the spectrum of mutants recovered (i.e., the number of complementary
classes) and the overall frequency of independently induced zym mutations. In
both respects, UV mutagenesis appeared to be more effective than treatment
with EMS. All zym mutants recovered after EMS mutagenesis proved to be
members of the same complementation group, whereas those isolated after UV
mutagenesis defined seven complementation groups. However, even in the
latter case, independently arising mutations within the complementation group
defined by the EMS-induced mutants were common.
The apparent site-specific mutagenesis obtained after EMS treatment cannot,
however, be directly correlated to the choice of mutagen because the two
mutant searches differed in other details of experimental protocol as well. In
particular, the EMS mutagenesis was performed on vegetative cells, whereas
UV mutagenesis was conducted on cells grown for 18 hr in mating-induction
medium and thus undergoing gametic differentiation. Furthermore, different
strains were utilized in the two experiments (see Figure 1).Preliminary results
from UV mutagenesis of a third strain (the wild-type C. monoica strain maintained by The University of Texas Culture Collection of Algae) suggest that
additional zym loci (i.e., new complementation groups not described here) exist
and that use of vegetative cells does not preclude isolation of mutants from
several complementation groups, nor does it necessitate excessive recovery of
mutants within the cistron defined by the original EMS mutagenesis. Thus the
peculiar results reported here may be specific to EMS mutagenesis and/or to
the particular strain utilized.
Our initial screening method for the identification of zym mutants involved
visual inspection of cultures by light microscopy and relied, first of all, upon
the absence of the discarded primary zygote wall in mated cultures. Thus we
have selected for mutants with defects expressed relatively early in the maturation process. In renewed mutant searches (in progress), we are now using
chloroform selection as the first step in our screening process; preliminary
results indicate that mutants with maturation defects expressed after primary
zygote wall release (but before induction of germination) can also be readily
obtained from C. monoica.
The zym markers identified thus far are expressed only in the zygote and act
there as recessive lethal mutations. Thus they have proved invaluable for the
selection of heterozygous zygotes from the background of self-matings characteristic of homothallic strains and typically hampering genetic analyses. Incorporation of the zym markers into strains carrying other mutations of interestsuch as the mutations to antibiotic resistance (VANWINKLE-SWIFT
1979, 1980),
auxotrophy (VANWINKLE-SWIFT
1979), or self-sterility (VANWINKLE-SWIFT
and
BAUER1982) previously isolated in our laboratory-will greatly facilitate our
efforts to map the nuclear genome of this species. However, because production
of a normal viable zygote in crosses between zym strains requires the use of
complementary zym parents, we cannot directly assess the linkage relationships
between independently isolated zym markers within a given complementation
group by standard tests for allelism.
442
K. P. VANWINKLE-SWIFT AND C. G . BURRASCANO
Successful completion of the Chlamydomonas sexual cycle involves a complex progression of cellular events that must occur in an ordered sequence.
Thus, for example, cell fusion must occur before nuclear fusion, and nuclear
fusion before the induction of meiosis. Similarly, zygospore wall development
is restricted to the product of mating events and does not occur in the haploid
gametes. We have considered the possibility that mating and zygote maturation
may be coordinately regulated in a complex fashion, and that such coordinate
regulation could lead to a misinterpretation of complementation data for zygote
maturation genes.
Suppose, for example, that each cell from a homothallic species such as C.
monoica carries copies of two mating-type “genes” analogous to the mt+ and
mt- mating-type alleles of heterothallic species, and that pair formation in a
clonal culture results from the expression of the mt+ locus in some cells and the
mt- locus in others (a situation similar to that described for homothallic strains
of the yeast Saccharomyces cerevisiae; HERSKOWITZ
et al. 1980; cf. VANWINKLESWIFTand BAUER1982 for further discussion). Suppose also that these loci are
regulatory and control the expression of structural genes-some of which are
required for a particular mating behavior and others of which take part in
zygote maturation. Cells expressing the mt+ gene are hypothesized to produce
one set of zygote maturation gene products whereas cells expressing the mtlocus produce a second set. Maturation per se might require the interaction of
these two subsets of gene products and would thus begin only after fusion of
the mt+ and mt- -acting gametes. If such a situation were to exist, an absence
of complementation between two zym markers could occur for mutations in
separate genes which are exclusively expressed in the same mating-type-these
mutations need not be physically linked. Table 5 summarizes the effects of
various types of coordinate regulation on the outcome of a standard complementation test. The failure of two point mutations to complement one another
may not necessarily indicate allelism and does not necessarily imply dominance
of the mutant allele(s); instead it may be a consequence of similar sex-limited
expression for the gene loci involved (model I, Table 5). Note also that a positive
complementation test could be obtained even if one of the mutations involved
were “dominant” to its wild-type allele (models 11, 111, Table 5 ) . In fact, at
present we have no means of accurately assessing dominance if a maturation
marker shows mating-type-specific expression, because the wild-type and mutant alleles would never be jointly expressed in the zygote.
Our recovery of more than two zym complementation groups suggests that
not all maturation genes show “mating-type-specific’’ expression, but the possibility remains that one or more of the zym loci identified thus far could be
coordinately expressed with “mating-type”. In the yeast S. cerevisiae, the
mating-type alleles (MATa and MATa) not only determine haploid mating
behavior, but are also involved in the control of meiosis and sporulation in
diploids produced by MATa X MATa matings (KASSIRand SIMCHEN
1976; KLAR,
FOGEL
and RADIN1979; HERSKOWITZ
et al. 1980; SPRAGUE,
RINEand HERSKOWITZ
1981). The existence of similarly complex loci in C. monoica might allow the
recovery of mutants with mating-type-specific defects that allow normal haploid
443
MATURATION MUTANTS OF C. MONOICA
TABLE 5
The effect of coordinate regulation of mating and zygote maturation on the
results obtained from a stondord complementation test"
Genotypes
Haploid gamete
types
Model
Strain
mt+
ZymA
ZymB
AB'
A+B
ZymA
ZymB
(111)
(IV)
mt-
Diploid zygotes produced by
Selfing
Crossingh
Apparent
complementation
~
(I)
(11)
A B+/A+ B/-
-
--
A B+
A+B
- B+
-B
A B+/A+ B/-
Bt
B
?4 A B+/-
ZymA
ZymB
AA+-
- Bt
-B
A -/A' -/-
B+
B
ZymA
ZymB
AB+
A+B
AB+
A+B
--e
-
A B+/A B+
A+ B/A+ B
- (Z)
- (Z)
No
No
% A+ B/-
B (Z)
B+ (WT)
No
Yesd
?hA -/?4A+ -/-
B (Z)
B+ (WT)
No
Yes
'h A B+/?hA'
B/-
A+ B/A B+ (WT)
Yesd
a Main assumptions: (a) Homothallic strains differentiate into two gamete types analogous to the
mt+ and mt- mating types of heterothallic species; (b) Certain genes required for zygote maturation
may be specifically induced (expressed) in one gamete type only. Alternative models: (I) Zym genes
A and B are both induced only in mt+-acting gametes; (11) ZymA is mt+-specific but zymB is
expressed in both gamete types; (111) ZymA is mt+-specific and zymB is mt--specific; ( I V ) ZymA
and zymB are both expressed in both gamete types.
'Phenotypes of zygotes produced by inter-strain matings are shown in parentheses: Z =
maturation-defective: WT = normal, fully matured zygotes.
= Unexpressed gene locus.
Mutant alleles at non-mating-type-specific loci are assumed to be recessive.
-
mating but interfere with zygote maturation. Thus it is also conceivable that
some of our zym markers might reside at one or the other of the hypothesized
"mating-type loci".
As mentioned previously, such speculation cannot be directly evaluated by
allelism testing of noncomplementing mutants since pairing between such zym
strains does not produce viable zygotes. However, less direct approaches are
feasible and might include the identification of nonselective markers closely
linked to a given zym marker that could then be mapped to other zym markers
within the same complementation group, or the isolation of conditional zym
mutants or zym suppressor mutations that would allow the recovery of viable
zygotes from crosses between noncomplementary maturation-defective strains.
Continued genetic analysis of maturation mutants, and the extension of the
work to include ultrastructural and biochemical studies, should provide clues to
the underlying molecular mechanisms that insure progression of zygote development from initial cell fusion through full development of the mature zygospore.
We would like to thank Drs. C. A. BARNETT,
R. L. WEISSand F. AWBREY
for providing access to
needed laboratory space and equipment, and Drs. A. BAERand D. SHORTfor additional research
support. K. V.W.-S. wishes to thank Dr. R. A. LEWINfor first introducing her to Chlamydomonas
444
K . P. VANWINKLE-SWIFT AND C. G. BURRASCANO
monoica and for encouragement and helpful discussions throughout the course of this work. This
study was made possible by a grant from the National Science Foundation (PCM-8010026) to
K.V.W.-S.
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Corresponding editor: J. E. BOYNTON